Cellular organelles such as mitochondria carry genetic information for coding only a limited number of protein components. Thus, the majority of mitochondrial proteins are coded in the nucleus and synthesized in the cytoplasm. One of the fundamental questions of mitochondrial biogenesis is how cytoplasmically synthesized proteins cross the existing outer and inner membrane and are incorporated into the mitochondrion. To clarify this question, the biosynthesis of carbamylphosphate synthetase and ornithine transcarbamylase, two enzymes of the mitochondrial matrix, will be investigated. The kinetics of enzyme synthesis will be studied to determine whether the transport of cytoplasmically synthesized proteins occurs as a post-translational event or by vectorial translation. The site of synthesis of the enzymes will be localized by immunochemical methods. Attempts will be made to obtain putative precursors of carbamylphosphate synthetase and ornithine transcarbamylase. The polypeptide products of in vivo synthesis and the translation products of in vitro synthesis utilizing heterologous protein synthesizng systems will be isolated by immunoprecipitation and chemically characterized.